Real-Time Neurotransmitter Sensing in Midbrain Organoids Using 3D Carbon Cup Electrodes

Real-time, chemically selective detection of neurotransmitter release in brain organoids remains a challenge due to their complex three-dimensional architecture and limited optical accessibility. We introduce 3D-printed carbon cup electrodes (CCEs) -pyrolyzed, multi-level pillar structures - designed to confine a single organoid, increase electroactive surface area, and enable amperometric recordings of oxidizable neurotransmitters. The CCEs exhibit excellent electrochemical performance and reproducibility, supporting diffusion-controlled dopamine redox behavior without interference from culture medium. Using ventral midbrain (vMB) organoids at two maturation stages (Day 30 and Day 90), we demonstrate real-time detection of potassium-evoked dopamine release directly from organoids. Mature organoids exhibit higher peak currents, correlating with increased tyrosine hydroxylase (TH⁺) expression confirmed by immunocytochemistry. Embedding organoids in Matrigel significantly attenuates dopamine signals, indicating a diffusion barrier that limits stimulant access and/or dopamine transport to the electrode. Repetitive stimulations reveal progressive depletion of dopamine stores, consistent with diminished vesicular release. Chemical perturbations using 6-hydroxydopamine and verapamil further validate the platform by producing substantial reductions in dopamine release accompanied by loss of TH⁺ neurons. These results establish CCEs as a powerful tool for label-free, real-time monitoring of neurotransmission in human organoids, with potential for multiplexed sensing and integration of enzymatic detection strategies for non-electroactive neurotransmitters.